Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease characterized by the destruction of the insulin producing cells found in the pancreatic islets of Langerhans. Impaired immunoregulation within the islets contributes to T1D in rodent models such as NOD mice, and very likely in humans. In NOD mice, onset of diabetes is marked by: i) heavy infiltration of the islets by pathogenic T cells and proinflammatory antigen presenting cells, ii) a diminished pool of islet Foxp3-expressing immunoregulatory T cells (Foxp3+Treg), and iii) the loss of 80-90% of cell mass. The nature and effectors of islet inflammation in human T1D appear to be more variable. Studies have reported cadaveric T1D pancreases being heavily infiltrated with T cells, but subjects with significant residual cell mass and in some instances, no detectable islet infiltration, have als been observed. We propose that directly manipulating the islet inflammatory milieu will prove to be the most effective strategy to broadly treat subsets of T1D. Recently, we demonstrated that late preclinical T1D is suppressed in NOD mice by targeting IL-2 expression to cells in vivo via adeno-associated virus (AAV) vector gene delivery. Protection was due to islet-specific expansion of Foxp3+Treg with enhanced suppressor function. Importantly, IL-2 expression was localized to the islets thereby avoiding the unwanted complications associated with systemic delivery of a potent, pleiotropic cytokine such as IL-2. The current application proposes to use AAV vectors to co-express anti-inflammatory cytokines in the islets to promote a synergistic effect leading to robust immunoregulation. Aim 1 will focus on defining mechanisms of synergy induced via combinatorial cell-specific cytokine expression in recent onset diabetic NOD mice. Aim 2 will explore the in vivo effects of ectopic cytokine expression on tissue-resident human effector T cells and FOXP3+Treg using humanized mice. A human islet allograft model is also being exploited to directly establish the efficacy of cell-specific cytokine expression on suppressing human islet pathology. The underlying hypothesis for this proposal is that Foxp3+Treg are regulated by non-redundant cytokine signals that together act synergistically to enhance homeostasis, fitness and function. Similarly, multiple cytokine signaling events synergize to mediate distinct mechanisms of Teff tolerance. Therefore combining anti-inflammatory cytokines for the purpose of immunotherapy will induce superior and qualitatively distinct immunoregulation. This proposal will advance our general understanding of how cytokines interact to regulate Foxp3+Treg immunobiology and Teff pathogenicity.